1H, 13C and 15N NMR backbone assignments and secondary structure of the 269-residue protease subtilisin 309 from Bacillus lentus

Abstract

1H, 13C and 15N NMR assignments of the backbone atoms of subtilisin 309, secreted by Bacillus lentus, have been made using heteronuclear 3D NMR techniques. With 269 amino acids, this protein is one of the largest proteins to be sequentially assigned by NMR methods to date. Because of the size of the protein, some useful 3D correlation experiments were too insensitive to be used in the procedure. The HNCO, HN(CO)-CA, HNCA and HCACO experiments are robust enough to provide most of the expected correlations for a protein of this size. It was necessary to use several experiments to unambiguously determine a majority of the alpha-protons. Combined use of HCACO, HN(COCA)HA, HN(CA)HA, 15N TOCSY-HMQC and 15N NOESY-HMQC experiments provided the H alpha chemical shifts. Correlations for glycine protons were absent from most of the spectra. A combination of automated and interactive steps was used in the process, similar to that outlined by Ikura et al. [(1990) J. Am. Chem. Soc., 112, 9020-9022] in the seminal paper on heteronuclear backbone assignment. A major impediment to the linking process was the amount of overlap in the C alpha and H alpha frequencies. Ambiguities resulting from this redundancy were solved primarily by assignment of amino acid type, using C alpha chemical shifts and 'TOCSY ladders'. Ninety-four percent of the backbone resonances are reported for this subtilisin. The secondary structure was analyzed using 3D 15N NOESY-HMQC data and C alpha secondary chemical shifts. Comparison with the X-ray structure [Betzel et al. (1992) J. Mol. Biol., 223, 427-445] shows no major differences.

1H, 13C, and 15N resonance assignments of Fusarium solani pisi cutinase and preliminary features of the structure in solution

“…Instead, water suppression was typically done by applying a proton spin-lock pulse (Messerle et al, 1989) after the first INEPT transfer. A slightly modified version of the 3D CO-CAH experiment (Dijkstra et al, 1994) was recorded on the H 2 0 sample, to prevent interpretation problems due to deuterium isotope shifts and variation in sample conditions (Remerowski et al, 1994;Zhang & Gmeiner, 1996), as well as on the D 2 0 sample. CO and C" frequency labeling was performed in a constant-time fashion.…”

Solution Structure of the 30 kDa N-Terminal Domain of Enzyme I of the Escherichia coli Phosphoenolpyruvate:Sugar Phosphotransferase System by Multidimensional NMR

“…Backbone assignments using conventional triple resonance experiments on 15 N/ 13 Clabeled samples have also been obtained for single chain monomeric proteins up to 269 residues, in particular on two ∼27 kDa members of the subtilisin family of proteases (Fogh et al, 1994;Remerowski et al, 1994). No three-dimensional solution NMR structure, however, of a protein much larger than about 200 residues has yet been published, and thus EIN, at 259 residues in length, is clearly among the largest monomeric proteins whose three-dimensional structure is being solved at the present time by NMR.…”

A Soluble Major Histocompatibility Complex Class I Peptide-binding Platform Undergoes a Conformational Change in Response to Peptide Epitopes

“…Our results show that this is indeed the case and open up the possibility of studying the conformational change by conventional biophysical techniques. Indeed, D 1-193 b is of a size which is compatible with two-dimensional NMR spectral analysis (22,23).…”